A number of structural studies have confirmed the binding of sigma54 to RNAP and DNA. Sigma54 binds DNA -24 and -12 base pairs from the transcription start site to initiate DNA melting, an essential step in transcription. A substantial body of work, including DNA footprinting, alanine scanning mutation studies, low resolution cryoelectron microscopy of sigma54-RNA-polyermase bound to DNA in the closed configuration, and high resolution NMR structure has shown that sigma54 binds specifically and tightly to the major groove of DNA. For example the Wemmer lab used NMR to show that the 66 amino acid long helix-turn-helix (HTH) motif from in Aquifex aeolicus binds DNA. Within this C-terminal HTH motif, a single alpha-helix at residues 377 to 386 (ARRTVAKYRE), termed the RpoN box, is responsible for binding to the major groove of DNA (PBD: 2O8K). Replacement of Arg378, Arg379, Tyr384 and Arg385 with Ala decreased DNA binding substantially. The protein NMR structure shows that this helix interacts selectively with the -24 region (5'-TGGCACG-3') of the promoter. In particular, Arg378 and Arg379 are localized to the -24 element of the interaction and make multiple hydrogen bonding and ionic interactions with Gua-25 and Gua-26 on the non-coding strand of DNA based on significant line-broadening of a 15N-HSQC spectrum. This leads to a high affinity interaction between the 66-mer HTH motif and the promoter region (Kd = 114 nM) Although the peptides are modeled to inhibit transcription, it is necessary to assess cell viability to ensure that downstream antivirulence effects are observable. By using the standard broth microdilution method,24 the antimicrobial activity of all stapled peptide analogs was assessed. The minimum inhibitory concentration (MIC) value of all peptides is 32 microg/mL or higher. As this value is high compared to many conventional antibiotics, the peptide can be assessed at low concentrations to study its effects on virulence properties of Gram negative bacteria. To determine whether the compounds are cytotoxic to eukaryotic cells, WS1 fibroblasts were exposed to serial dilutions of each compound. The data demonstrate that the compounds are not toxic to normal cells over a concentration range peaking at 10 microM. In order to assess the helical structure of the synthesized peptides, circular dichroism spectroscopy was carried out by dissolving the compounds in water. While the wild type sigma54 peptide is unstructured, stapled peptides 1-4 all possess the hallmark spectrum of an alpha-helical secondary structure. Flow cytometry was used to gain a high-throughput perspective on the effectiveness of each stapled peptide in a large population of bacteria. By making use of its individual event detection, the percentage of cellular uptake of the peptides was determined. In both E. coli and P. aeruginosa species of Gram negative bacteria, all four peptide analogs are capable of penetration. Compared to the vehicle control, the wild type peptide, the sequence in which no staple is present, shows minor penetration in PA01 P. aeruginosa and insignificant levels of penetration in BW25113 E. coli. With each peptide, there is a penetration of at least 50% of cell in each peptide sample. In particular, sigma54-2 appears to have the highest penetrance whereas sigma54-4 has the least. To determine the mode of transport within the cell, sodium azide was used to inactivate ATPases used in hydrogen ion transport. All interior transport will be conducted through a non-active method such as passive or facilitated diffusion. With both types of treatments, it's possible to determine whether the mode of uptake of stapled peptides is through active or passive transport. Furthermore, with passive transport, the entry of the peptide should be faster than active due to the lack of metabolic means necessary to promote cell entry. The majority of peptide analogs increase in penetration upon sodium azide treatment. This may be an indicator that the peptide is able to be pumped out by bacteria to a degree. Overall, this assay demonstrates that stapled peptides are capable of penetrating cells better than their unstapled counterpart in a charge-independent manner. Further studies in confocal microscopy were conducted to gain a low-throughput visual method of assessing cell penetrance. The peptides correlate with the flow cytometry data in that some cells display strong fluorescence intensity and others display less. By using an image-based detection method for permeability, we observe that the double-stranded DNA of E. coli appears to aggregate in the center away from the membrane. In many of the cells observed, a number of the cells were undergoing cell division but still had significant uptake of these peptides. With the cell membranes, it is seen that the peptide does not integrate itself into the membrane due to a lack of green or masking of the red membrane stain. These peptides must cross both the cell wall and cell membrane to remain in the cytoplasm of bacteria. The binding of sigma54 RpoN with the -24 site of glnA was thoroughly examined through the use of fluorescence anisotropy coupled with fluorescence energy transfer. Fluorescence anisotropy is a powerful tool in determining biomolecular affinities where the emission polarization and the molecular rotational diffusion are dependent upon the molecular binding. In this study, the change in anisotropy of FITC tagged sigma54 stapled alpha-helical peptides at varying concentrations of the double stranded -24 glnA sequence were studied thoroughly to determine the dissociation constant (kD) between the peptide and the DNA. Next, we used the fluorescence lifetime imaging microscope (FLIM) setup in order to evaluate the specific binding between the peptide analogs and the double-stranded glnA DNA sequence of 30 base pairs in length bound to a solid phase. Foester resonance energy transfer (FRET) is a widely used technique in determining biomolecular binding along with the quantitative analysis of biomolecular conformational and structural changes where FRET is observed upon the close proximity of the donor and acceptor fluorophores (1-10 nm). FITC was selected as the peptide donor due to its excitation at 488 nm and Cy5 as the acceptor due to its lack of 488 nm excitation, its absorption within FITC's emission range, and its emission outside the FITC emission range. In this experimental setup, a Cy5 tagged DNA (acceptor) was immobilized on to a thin film of gold nanoparticles (AuNP), where the latter's purpose is to enhance the single molecular fluorescence events. By using single molecular events, it is possible to visualize the direct interaction between DNA and peptide. FITC tagged peptide (donor) was then added to the AuNP-glnA conjugates, with FRET only to be observed upon the specific binding of the DNA and the stapled peptide. As the maxima of both tagged and non-tagged FITC are aligned, the interaction between DNA and peptide displays a profile that shows FRET occurs due to the presence of a Cy5 signal from an excitation at 488 nm. A modified classical motility assay was used to determine the bacteria's ability to travel through soft media. Knockout MC4100 strain displays a phenotype in which the bacteria do not grow significantly away from the point of inoculation. In BW25113 cells, all peptides show a degree of inhibition relative to the vehicle and wild-type peptide. As demonstrated from the change in diameter of bacterial growth, it is shown that the peptides do not possess same motility inhibition compared to a knocked out cell line. As the peptide decreases the expression of motility-related genes, the overall ability of the peptides does demonstrate a decrease in flagellar mobility of the bacteria.